Missense RHD single nucleotide variants induce weakened D antigen expression by altering splicing and/or protein expression

Background Although D variant phenotype is known to be due genetic defects, including rare missense single nucleotide variants (SNVs), within the RHD gene, few studies have addressed molecular and cellular mechanisms driving this altered expression. We others showed previously that splicing commonly disrupted by SNVs in constitutive splice sites their vicinity. thus sought investigate whether located “deep” exonic regions could also impair mechanism. Study design methods Forty-six reported exons 6 7 were first selected from Human RhesusBase. Their respective effect on was assessed using an vitro assay. An RhD-negative cell model further generated CRISPR-Cas9 approach. RhD-mutated proteins overexpressed newly created model, membrane expression of antigen measured flow cytometry. Results Minigene assay 14 46 (30.4%) alter splicing. Very interestingly, investigation two SNVs, which both affect codon 338 confer a weak phenotype, various mechanisms: c.1012C>G (p.Leu338Val) disrupts only, while c.1013T>C (p.Leu338Pro) alters only protein structure, agreement with silico prediction tools 3D structure visualization. Conclusion Our functional data set suggests damage quantitatively by, at least, different (splicing alteration destabilization) may act independently. These thereby contribute extend current knowledge governing weakened